• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1761-1767
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• 2015

Sevenband grouper, Epinephelus septemfasciatus, is becoming an important aquaculture species in Korea. However, viral nervous necrosis disease is a large problem causing mass mortality in sevenband grouper aquaculture. Recombinant protein vaccines are one of the best methods to reduce these economic losses. However, the cell-based expression method mainly produces inclusion bodies and requires additional procedures. In this study, we expressed a recombinant viral coat protein of sevenband grouper nervous necrosis virus (NNV) using a cell-free protein synthesis system. The purified recombinant NNV coat protein (rNNV-CP) was injected into sevenband grouper at different doses followed by a NNV challenge. Nonimmunized fish in the first trial (20 μg/fish) began to die 5 days post-challenge and reached 70% cumulative mortality. In contrast, immunized fish also starting dying 5 days postchallenge but lower cumulative mortality (10%) was observed. Cumulative morality in the second trial with different doses (20, 4, and 0.8 μg/fish) was 10%, 40%, and 50%, respectively. These results suggest that rNNV-CP can effectively immunize sevenband grouper depending on the dose administered. This study provides a new approach to develop a recombinant vaccine against NNV infection for sevenband grouper.

• Journal of fish pathology
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• v.34 no.1
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• pp.99-104
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• 2021

In this study, we investigated safety and efficacy of a low temperature immunization protocol with NNV in red spotted grouper, Epinephelus akaara and long tooth grouper, Epinephelus bruneus and seven band grouper, Hyporthodus septemfasciatus. Further, growth rate between immunized and naïve fish was evaluated during the experiment to check side effect of immunization. Three grouper species were immunized by immersion method with live NNV at 10^5.0 TCID₅₀/mL at 16.5℃ for 30 min and reared for 120 days at natural sea water temperature. To evaluate growth rate, total length and wet weight was measured 7 times after immunization. Immunized three grouper species were challenged by intramuscular inoculation with NNV at 10^4.2 TCID₅₀/100 µL/fish. Immunization at low temperature with live NNV did not show any clinical symptoms of infection, mortality and inhibition of growth. After challenge, cumulative mortality of naïve seven band grouper, red spotted grouper, long tooth grouper were 45, 10, 20 %, respectively. However no mortality was observed at immunized groupers. Thus, it was demonstrated that immunization at low temperature with live NNV are able to protect three different species of groupers without inhibition of growth.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• Fisheries and Aquatic Sciences
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• v.22 no.12
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• pp.28.1-28.8
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• 2019

Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.3
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• pp.127-132
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• 2007

The innate immune response which is seen as the initial defense mechanism induced upon foreign invasion has been well documented in higher vertebrates. This has also been observed in fish infected with NNV. However, the fish immune system based on fully established genome project has not been fully elucidated. Therefore, in this review, we hope to correlate NNV infection in fish that has devastated the aquaculture industry, to its host immune system. Further, we discuss the potential preventive measures in overcoming the widespread of this neurodisease.

• Korean Journal of Ichthyology
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• v.27 no.3
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• pp.183-188
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• 2015

Recently, mariculture of rainbow trout (Oncorhynchus mykiss) has been initiated in the coast areas of Korea. In the present study, we investigated the effect of fish viruses on mariculture of rainbow trout. The pathogenicity of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) isolated from freshwater rainbow trout was tested against major cultured marine fish species, including olive flounder (Paralichtys olivaceus), rock fish (Sebastes schlegeli), rock bream (Oplegnathus fasciatus), red seabream (Pagrus major) and sevenband grouper (Epinephelus septemfasciatus). The pathogenicity of marine birnavirus (MABV), hirame rhabdovirus (HIRRV) and nervous necrosis virus (NNV) isolated from marine fish species was also tested against rainbow trout. No mortality was observed in marine fish species challenged with IHNV or IPNV. However, olive flounder and rock bream were infected by IHNV and IPNV. A mortality of 8.3% was observed in rainbow trout challenged with HIRRV. The fish was infected by both MABV and NNV. These results suggest that the mariculture of rainbow trout might be affected by fish viruses.

• Journal of fish pathology
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• v.37 no.1
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• pp.1-8
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• 2024

The advantages of replicon vectors of RNA viruses include a high ability to stimulate innate immunity and exponential amplification of target mRNA leading to high expression of foreign antigens. The present study aimed to construct a DNA-layered nervous necrosis virus (NNV) replicon vector system in which the capsid protein gene was replaced with a foreign antigen gene and to compare the efficiency of foreign antigen expression between the conventional DNA vaccine vector and the present replicon vector. We presented the first report of a nodavirus DNA replicon-based foreign antigen expression system. Instead of a two-vector system, we devised a one-vector system containing both an NNV RNA-dependent RNA polymerase cassette and a foreign antigen-expressing cassette. This single-vector approach circumvents the issue of low foreign protein expression associated with the low co-transfection efficiency of a two-vector system. Cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 (with the capsid gene ORF replaced with VHSV glycoprotein ORF) exhibited significantly higher transcription of the VHSV glycoprotein gene compared to cells transfected with either a vector without hammerhead ribozyme or a conventional DNA vaccine vector expressing the VHSV glycoprotein. Furthermore, the transcription level of the VHSV glycoprotein in cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 showed a significant increase over time. These results suggest that NNV genome-based DNA replicon vectors have the potential to induce stronger and longer expression of target antigens compared to conventional DNA vaccine vectors.

• Journal of fish pathology
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• v.35 no.1
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• pp.121-128
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• 2022

Recent studies revealed that histone proteins are involved in innate immune responses during pathogen invasion as well as DNA packing. This study characterized the histone genes (H2A.V) of sevenband groupers and analyzed gene expression in NNV-infected sevenband groupers. The open reading frame (ORF) of H2A.V is 387 bp which encoded 128 amino acid residues. The deduced amino acid sequence of H2A.V harbor a highly conserved domain for H2A/H2B/H3 and H2A_C binding domain. Quantitative real-time PCR analysis showed that H2A.V had a high gene expression level in the brain and blood after being NNV-infected. An increase in extracellular histone protein in the blood has been identified as a biomarker for vascular function in humans. More research is required to understand histone's immune response at the protein level or in aquatic animals.

• Korean Journal of Ichthyology
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• v.29 no.3
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• pp.157-164
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• 2017

In 2015, a nervous necrosis virus (NNV) was isolated from sevenband grouper, Epinephelus septemfasciatus, maintained in land-based aquaculture system at below $12^{\circ}C$ in winter. Mortality was up to 30% in brood fish, over 4 kg of body weight. Moribund fish showed clinical sings typical of viral encephalopathy and retinopathy (VER), also called viral nervous necrosis (VNN), such as uncoordinated, corkscrew-like swimming behavior, belly-up at rest, darkening of body, cloudy eyeball and hyperinflation of the swim bladder. Aetiology of the disease was confirmed by gross observation of clinical signs, histopathology and molecular diagnosis. Histological studies revealed severe vacuolation and necrosis in the brain. Molecular diagnosis by revere transcription-polymerase chain reaction (RT-PCR) specific to batanodavirus yielded a positive result. The nucleotide sequences of the PCR-amplified fragment were 99.48~100% similar to barfin flounder nervous necrosis virus (BFNNV) genotype and most closely aligned with Pacific cod betanodavirus (PCNNV). This is the first report of natural batanodavirus, NNV infection in sevenband grouper reared in low water temperature during winter (below $12^{\circ}C$) in Korea.

• Journal of fish pathology
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• v.31 no.2
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• pp.71-79
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• 2018

Wild walleye pollock were caught from Goseong, The East Sea of Korea and examined for the existence of several fish pathogenic viruses; viral hemorrhagic septicemia virus (VHSV), nervous necrosis virus (NNV) and marine birnavirus (MABV). We collected 1,253 wild walleye pollock in total during February 2015 and August 2018. 324 spleen sample sets and 259 brain sample sets were made, and examined for the existence of the viruses mentioned above by reverse transcriptase polymerase chain reaction (RT-PCR). None of the target viruses were detected by one-step PCR. When some of these samples were further examined by two-step PCR, 19.7% (36/183) of spleen sample sets were positive for VHSV, and 4.4% (8/183) of spleen sample sets and 1.2% (3/259) of brain sample sets were positive for NNV. The target sequences of these viruses were clustered with those previously reported in Korea (Genotype IVa of VHSV, RGNNV genotype of NNV) by phylogenetic analysis. The activity of these viruses are not clear because virus isolation was not attempted, but probably very low because all the positive samples were detected by two-step PCR.